KR20190037546A - Composition for treating wound containing epidermal stem cell conditioned medium and use thereof - Google Patents

Abstract

Provided are a composition for treating wounds, which contains a culture medium of stem cell-derived skin precursor cells, a method for manufacturing the same, and a use thereof. According to the composition for treating wounds according to an aspect, it is possible to promote skin regeneration of an individual. According to the method for manufacturing the pharmaceutical composition for treating wounds according to an aspect, it is possible to promote wound healing of the individual.

Description

[0001] The present invention relates to a pharmaceutical composition comprising a dermal stem cell culture liquid and a use thereof.

A stem cell-derived skin precursor cell culture, a method for producing the same, and uses thereof.

The skin acts as a shield to protect the body from the outside. When a wound occurs in the skin, the wound is filled with blood by the natural healing action of the body, and the granule of the platelet is reduced, and the activation of the hageman factor starts and the wound healing process proceeds. Coagulation of blood is a temporary defensive action that protects exposed wound tissues and provides a basis for cells to move during the healing process.

Wound healing and / or healing occurs in three major stages. The first step is an inflammatory phase characterized by inflammation at the traumatic site. Inflammation can be generally short-lived in the absence of significant infection. The second stage is the proliferative phase, which is characterized by epithelialization, angiogenesis, granulation tissue formation and collagen deposition. Angiogenesis with new capillary formation is used to deliver nutrients and maintain granulation tissue formation. Without the formation of new capillaries into the wound, essential nutrients do not reach the wound, creating a wound that is not chronically healed. The surface of the injured wound is covered by a layer of keratinocytes, resulting in new epidermis and re-epithelialization where the epithelial layer is reconstructed. When the re-epithelization is completed, a series of processes are performed in which the wound area is reduced through an increase in connective tissue and a reorganization. The final stage of wound healing is the maturational phase in which fibroblasts differentiate into collagen. During maturation, the junk cells and capillaries of the convalescent tissue gradually disappear, and scarring can occur if the cells and capillaries of these tissues are hyperplasia or not normally degraded.

On the other hand, the skin consists of the epidermis, the dermis, and the subcutaneous fat layer in order. The epidermis is divided into stratum corneum, granular layer, superficial layer and basal layer in order from the outside. Cells of the epidermal layer act like bricks, Acts like a mortar to form a skin barrier. The dermis is composed of fibroblasts and produces proteins such as collagen and elastin and mucopolysaccharide to form extra cellular matrix (ECM). Through proper decomposition, absorption and remodeling of tissues It happens.

Since it is important not only to treat wounds quickly in the course of wound healing but also to treat them without side effects or scars, efforts are being made to find substances that have the potential to promote collagen production.

One aspect provides a pharmaceutical composition for treating a wound comprising a stem cell-derived skin precursor cell culture liquid.

Another aspect provides a method of making a pharmaceutical composition for wound healing.

Another aspect provides a method of treating a wound in a subject comprising administering to the subject a composition as described above.

One aspect provides a pharmaceutical composition for treating a wound comprising a stem cell-derived skin precursor cell culture liquid.

The pharmaceutical composition may be for treating a wound. The composition may be one having skin regeneration effect. The pharmaceutical composition may be one for promoting the formation or regeneration of dermal tissue.

The term " wound " refers to a disorder in which tissue is cut, torn, broken, burned, traumatized, Or injury to the human body resulting from the disease. The term " tissue " refers to a mass of cells in the body that together form a particular function. The tissue may be an eye, mucus, lung, kidney, heart, visceral, tendon, vascular tissue, bone, skin, connective tissue, and nerves such as spinal cord. The wound may be an open wound whose surface is open, or a closed wound whose surface is not open. An example of such a wound may be an open wound in the skin.

An example of such a wound is the epidermis of the skin; Dermis; Epidermis and dermis; Or the damaged skin of the epidermis, dermis and subcutaneous fat layer.

Examples of such wounds include but are not limited to cuts, incisions (e.g., surgical incisions), wound, abrasions, contusions, stab wounds, raises, lacerations or lacerations, fractures ), Burns, or amputations.

The wound may be lesion, sore, necrosis, and ulcer. The necrosis may be associated with dead tissue resulting from infection, injury or infarction. The ulcer may be a local defect or dent on the surface of the organ or tissue produced by the dissection of the necrotic tissue. An example of the wound may be a foot ulcer and / or necrosis caused by a disease, specifically, a diabetic foot ulcer and / or necrosis.

The skin consists of the epidermis, the dermal layer and the subcutaneous fat layer. The basal layer is located at the lowest part of the epidermis, and new epidermal cells (keratinocyte, skin tissue or keratinocyte) are formed in the basal layer through cell division, and the epidermal cells formed form dead cells Continue to replace it. This process induces regeneration of skin cells. The stem cell-derived skin precursor cell may be a skin layer, for example, a cell constituting the basal layer. The stem cell-derived skin precursor cells may be precursor cells of epidermal cells. The stem cell-derived skin precursor cells may have a limited ability to differentiate from the stem cells in terms of their differentiation potential and self-renewal ability. The stem cell-derived skin precursor cells can be used interchangeably with stem cell-derived epidermal progenitor cells, differentiated skin precursor cells, differentiated epidermal cells, dermal stem cells or epidermal stem cells. The stem cell-derived skin precursor cells may be one in which at least one gene selected from the group consisting of keratin 5 (Krat 5), keratin 1 (Krt 1) and keratin 14 (Krat 14) is expressed. The stem cell-derived skin precursor cells may be one having at least one cell surface marker characteristic selected from the group consisting of keratin 5, keratin 1 and keratin 14. The stem cell-derived skin precursor cells may have increased expression of at least one selected from the group consisting of keratin 5, keratin 1 and keratin 14 as compared to stem cells. The stem cell-derived skin precursor cells may be differentiated into a state in which a large amount of Krt10 and / or IVL is not reached in the late stage of differentiation depending on the degree of differentiation. The expression can be confirmed by measuring changes in gene expression using techniques such as real-time PCR (RT-PCR) or immunoblot (IB). The stem cell-derived skin precursor cells may exhibit a specific shape of a circle of a certain size.

The stem cell-derived skin precursor cell culture solution may be a culture of stem cell-derived skin precursor cells obtained in the course of culturing the stem cell-derived skin precursor cells in a medium or a supernatant thereof. The stem cell-derived skin precursor cell culture liquid may be a culture of stem cell-derived skin precursor cells or a concentrate thereof or a lyophilized product thereof obtained during culturing or culturing of stem cell-derived skin precursor cells Lt; / RTI > The concentrate may be cultured for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 5 times the culture of stem cell-derived skin precursor cells or the supernatant thereof. About 10 times, or about 20 times. The stem cell-derived skin precursor cell culture solution may contain a cell-derived protein that secretes a useful protein through secretion of cells during cell culturing of the stem cell-derived skin precursor cell and secretes the protein out of the cell. The protein may be one comprising a useful protein such as a cytokine, a growth factor or the like. The stem cell-derived skin precursor cell culture solution may contain the protein shown in FIG. The stem cell-derived skin precursor cell culture solution contains at least one of Thrombospondin (TSP), Tissue inhibitor of metalloproteinases 1 (TIMP1), Tissue inhibitor of metalloproteinases 2 : TIMP2), Ectodysplasin-A2 (EDA-A2), X-linked ectodysplasin-A receptor (XEDAR), Angiopoietin -1, secreted protein acidic and rich in cysteine (SPARC), EGF-like and two Follistatin-1 (EGF-like and two Follistatin- -like domains 1 / Tomoregulin-1: TMEFF1 / Tomoregulin-1, Nidogen-1, Insulin-like growth factor-3 (IGFBP- , Thrombospondin-2, tumor necrosis factor-related activity induction cytokine It may be one containing at least one selected from the group consisting of a (TNF-related activation-induced cytokine:: TRANCE), and IL-15 receptor alpha (IL-15R alpha interleukin-15 receptor alpha). The stem cell-derived skin precursor cell culture solution contains at least one of Thrombospondin (TSP), Tissue inhibitor of metalloproteinases 2 (TIMP2), Tissue inhibitor of metalloproteinases 1 : TIMP1), Ectodysplasin-A2 (EDA-A2) X-linked ectodysplasin-A receptor (XEDAR), Angiopoietin- 1 and Transmembrane protein with EGF-like and two Follistatin-1 with secreted protein acidic and rich in cysteine (SPARC), epithelial growth factor-like and two polystatin- like growth factor-3 (IGFBP-3), insulin-like growth factor-3 (IGFBP-3) Thrombospondin-2, tumor necrosis factor-related activity-inducing cytokines At least one concentration selected from the group consisting of TNF-related activation-induced cytokine (TRANCE) and interleukin-15 receptor alpha (IL-15R alpha) is about 10 pg / Ml or more, or about 20 pg / ml or more.

The stem cell-derived skin precursor cell culture medium contains GROucho (GRO), latent TGF-beta binding protein 1 (latent TGF-beta bp1), Crossveinless- (Invitrogen-8, CV-2), Smad4, Interleukin-8 (IL-8), Activin C and Actin A .

The stem cell-derived skin precursor cell culture solution contains a CC chemokine receptor type 4 (CCR4), a granulocyte chemotactic protein-2 (GCP-2) / chemokine (CXC Motif ligand 6: CXCL6), endocan, P-selectin, CC chemokine receptor type 5 (CCR5), regulated 0.01% or less, or 0.005% or less, of at least one member selected from the group consisting of on Activation, ≪ / RTI > or not.

The composition may further comprise Epidermal Growth Factor (EGF).

The composition may include the stem cell-derived skin precursor cell culture liquid as an active ingredient or an effective ingredient. &Quot; Active ingredient or active ingredient " is intended to carry out the function referred to herein, except that it is included in minor amounts as impurities and thus does not perform the function.

The treatment may be to provide wound healing at a reduced time compared to natural healing. The treatment may include improvement and / or alleviation of the wound. Improvement of wound means lowering the degree of wound. In addition, the treatment may include treatment of a wound and / or a wound-related disease. Such treatment may mean healing and / or regeneration of damaged tissue caused by a wound. The wound treatment may include the meaning of skin regeneration. In addition, the treatment may be to maintain the original composition of the damaged tissue. In addition, the treatment may be to promote healing and / or regeneration of the damaged tissue while minimizing complications and / or scarring of wound related diseases.

The composition may be for promoting the pre-stage of the wound healing process. In addition, the composition may be one that promotes the proliferative and / or mature phase during the wound healing step. Thus, the composition may be for treating wounds in the proliferative and / or maturation phase.

The composition may include a " therapeutically effective amount " of the stem cell-derived skin precursor cell culture fluid. In this composition, " therapeutically effective amount " means an amount sufficient to exhibit a therapeutic effect when administered to a subject or a cell in need thereof. &Quot; Treatment " means treating a disease or medical condition in a mammal, including a human, including an individual, which includes: (a) preventing the occurrence of the disease or medical condition, cure; (b) relieving the disease or medical condition, i. e., eliminating or ameliorating the disease or medical condition in the patient; (c) inhibiting the disease or medical condition, i.e. slowing or stopping the progression of the disease or medical condition in the individual; Or (d) relieving the disease or medical condition in the subject.

The composition may be one that increases collagen synthesis or activity in cells.

The stem cell-derived skin precursor cell may be a stem cell differentiated.

The term " stem cell " means a cell having potency and self-renewal ability. Stem cells are divided into pluripotency, multipotency, or unipotency depending on the differentiation ability. The stem cells include embryonic stem cells (ESCs), adult stem cells (undifferentiated cells present in each tissue and organs), and stem cells (induced a pluripotent stem cell (iPSC) (a cell in which a gene and / or a protein is inserted into somatic cells to induce differentiation, or an induced pluripotent stem cell).

The stem cells may be mesenchymal stem cells. The term " mesenchymal stem cell (MSC) " is an adult stem cell, having multipotential and self-renewing ability, and excellent in proliferative capacity and genetically stabilized. The mesenchymal stem cells are cells that aid in the production of fat, cartilage, bone, bone marrow stroma, muscle, nerve, etc. and can differentiate into various cells such as adipocytes, cartilage cells, skin cells and bone cells . The types and origins of the stem cells are not limited as long as they have the ability to differentiate and self-regenerate. The stem cells may be derived from, for example, mammals, humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice or rabbits. The stem cells may be derived from isolated umbilical cord, placenta, fat, bone marrow, umbilical cord blood or amniotic fluid. The term " isolated " means that it is present in an environment different from that of a naturally occurring cell or tissue.

The term " umbilical cord " refers to a line connecting a mother and a belly so that a mammalian embryo can grow in the placenta. In general, there are three blood vessels surrounded by whitened jelly, i.e., two umbilical arteries and one umbilical vein As shown in FIG. The term " placenta " refers to an organ made for the fetus during mammalian pregnancy, with one side of the placenta contacting the mother and the other side abutting the fetus, It may be to supply nutrients. The placenta consists of amniotic membrane, chorion, and decidual membrane. The amniotic membrane is a thin, transparent membrane surrounding the fetus, which contains amniotic fluid, and amniotic fluid and / or amniotic membrane contains fetal stem cells. The decidual membrane is a membrane formed by the transformation of the uterine epithelial cells to be implanted in the uterus. There are stem cells of the mother. The chorionic membrane is the membrane between amniotic membrane and decidua surrounding fetal or amniotic fluid and is formed in the embryo and forms part of the striated membrane. Placenta-derived stem cells may be derived from the fetus or the mother. Amniotic stem cells may be derived from the fetus. Stem cells from the umbilical cord or the placenta are very abundant in their quantity and can proliferate and differentiate into other cells. The term " bone marrow " refers to tissues that make blood cells, including red blood cells, white blood cells, and platelets. The term " cord blood " refers to blood from the umbilical cord of a newborn after delivery. The term " fat " refers to body fat, and stem cells derived from fat are easy to obtain, and about 1% of adipocytes are estimated to be stem cells.

The stem cell-derived skin precursor cell culture liquid may contain about 0.01% to about 60% of the total volume of the pharmaceutical composition. The stem cell-derived skin precursor cell culture liquid may contain about 0.01% to about 60%, about 0.02% to about 50%, about 0.05% to about 30%, about 0.1% to about 20% From about 0.2% to about 19%, from about 0.4% to about 17%, from about 0.5% to about 16%, from about 0.6% to about 15% To about 12%, about 3% to about 10%, about 4% to about 7%, about 4% to about 6%, or about 5%.

The composition may comprise a pharmaceutically acceptable carrier. In this composition, " acceptable carrier " refers to a substance, generally an inert substance, used in combination with the active ingredient to aid in the application of the active ingredient. The carrier may be an excipient, a disintegrant, a binder, a lubricant, a diluent, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low substituted hydroxy cellulose, or a combination thereof. The disintegrant may be sodium starch glycolate, calcium monohydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or combinations thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The composition may be formulated into parenteral or oral dosage forms. The parenteral dosage form may be an injection or an external preparation for skin. The external skin preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof. The external preparation for skin may be a conventional external preparation for skin such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, an antioxidant, a surfactant, , Various skin nutrients, and the like can be appropriately compounded as needed. The external preparation for skin may be a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, fructose, fructose and other herbal medicines, various herbal medicines, tocopherol acetate, glycyrrhizic acid, Sugars such as trehalose and the like can also be appropriately compounded. Oral dosage forms may be tablets, capsules, aqueous solutions or suspensions. In the case of tablets, excipients such as lactose and corn starch, and lubricants such as magnesium stearate may be usually added. In the case of capsules, lactose and / or dried corn starch may be used as a diluent. When a suspending agent is required, the active ingredient may be combined with an emulsifying agent and / or a suspending agent. If desired, certain sweetening and / or flavoring agents may be added.

The stem cell-derived skin precursor cell culture solution may be prepared by culturing stem cells in a differentiation medium containing ascorbic acid and hydrocortisone to differentiate into stem cell-derived skin precursor cells; Culturing the differentiated stem cell-derived skin precursor cells in a medium to produce a culture of stem cell-derived skin precursor cells; And recovering the stem cell-derived precursor cell culture liquid from the culture of the stem cell-derived skin precursor cells. This step is described in detail in the following method.

In another aspect, the present invention provides a method for producing stem cells comprising culturing stem cells in a differentiation medium containing ascorbic acid and hydrocortisone to differentiate into stem cell-derived skin precursor cells; Culturing the differentiated stem cell-derived skin precursor cells in a medium to produce a culture of stem cell-derived skin precursor cells; And recovering the stem cell-derived precursor cell culture liquid from the culture of the stem cell-derived skin precursor cells. The present invention also provides a method for preparing a pharmaceutical composition for wound healing.

The method may include obtaining isolated umbilical cord, placenta, fat, bone marrow, umbilical cord blood or amniotic fluid. The separated umbilical cord, placenta, fat, bone marrow, umbilical cord blood or amniotic fluid can be obtained by conventional anatomical methods. The separated umbilical cord, placenta, amniotic fluid or umbilical cord blood may be umbilical cord, placenta, amniotic fluid or umbilical cord blood separated from the maternal body after delivery. The separated umbilical cord, placenta, amniotic fluid or umbilical cord blood may be stored in a sterilized container and ice after being separated. In the case of the placenta, for example, the placenta tissues present in the placenta can be obtained by cutting the placenta tissue into sterilized scissors. The umbilical cord may be obtained, for example, by separating the umbilical cord from the placenta and cutting it into sterilized scissors at various sites. The fat can be obtained, for example, by performing liposuction in the subcutaneous fat layer of the abdomen or thigh. The obtained placenta tissue, umbilical cord or fat is treated with phosphate buffered saline (PBS) containing antibiotics such as penicillin, streptomycin, gentamicin, or a combination thereof, once, twice or more than three times By washing, contaminants such as blood present in the tissue can be removed. The placenta, umbilical cord or fat can be reacted with the enzyme directly, or finely cut using sterilized scissors, and then the chopped cells can be reacted with the enzyme.

The method may comprise obtaining stem cells. The method of obtaining stem cells can be carried out by a method known to a person skilled in the art. In the case of umbilical cord or placenta, for example, the separated umbilical cord or placenta is attached to a culture vessel and cultured for 5 to 20 days, 10 to 20 days, or 10 to 15 days; Confirming that the cells extend from the cultured umbilical cord or the placenta; And / or reacting the umbilical cord or placenta with a cleavage enzyme. Or in the case of umbilical cord or placenta, for example, by reacting the isolated umbilical cord or placenta with a cleavage enzyme. The above-mentioned isolating enzyme may include collagenase. The collagenase refers to an enzyme that destroys the peptide bond of collagen, and may include collagenase type I, type II, type III, type IV, or a combination thereof. The separation enzyme is selected from the group consisting of 10 U / mL to 4000 U / mL, 20 U / mL to 2000 U / mL, 50 U / mL to 800 U / Ml, or 200 U / ml of collagenase. In addition, the isolating enzyme may include trypsin, Dispase or a combination thereof. In addition, the solution containing the above-mentioned isolating enzyme may include water, saline such as HBSS (Hank's Balanced Salt Solution) containing collagenase, trypsin, dyspazene or a combination thereof . The step of reacting the umbilical cord or placenta with the cleavage enzyme may be carried out by shaking or standing. The shaking or setting may be carried out at about 20 캜 to about 40 캜, about 30 캜 to about 40 캜, about 35 캜 to about 40 캜, or about 37 캜, and for 1 to 20 hours, To 9 hours, or 5 to 6 hours. In the case of fat, for example, it may comprise the step of reacting the isolated fat with a cleavage enzyme. The above-mentioned isolating enzyme may be a collagenase, trypsin, dyspazene or a combination thereof. The solution containing the cleavage enzyme may be water, saline, such as HBSS, including collagenase, trypsin, dyspazes, or a combination thereof. The step of reacting the fat with the separating enzyme may be carried out by shaking or standing.

In addition, after the placenta, umbilical cord or fat is allowed to react with a cleavage enzyme, a process for inactivating the cleavage enzyme may be performed. For example, serum may be added to stop the enzyme reaction. Methods for obtaining placental, umbilical cord or fat-derived stem cells after reacting the placenta, umbilical cord or fat with a cleavage enzyme can be performed by a method known to those skilled in the art. For example, after centrifuging, strainer) to separate cells.

The method may include culturing the obtained stem cells in a medium containing serum before the step of differentiating into stem cell-derived skin precursor cells. The serum may be fetal bovine serum (FBS), bovine calf serum (BCS), or a combination thereof. The serum may be from about 1% to about 50%, from about 2% to about 25%, from about 5% to about 20%, from about 7.5% to about 12.5%, or about 10% of the total volume of the differentiation medium . The medium may be one comprising fibroblast growth factor-4 (FGF-4) and / or heparin. The concentration of FGF-4 in the medium may be about 10 ng / ml to about 40 ng / ml, about 20 ng / ml to about 30 ng / ml, or about 25 ng / ml. The concentration of heparin in the medium may be about 0.5 μg / ml to about 2 μg / ml, about 0.5 μg / ml to about 1.5 μg / ml, or about 1 μg / ml. The medium may be one containing an antibiotic. The antibiotic may be one comprising penicillin, streptomycin, gentamicin, or a combination thereof. The concentration of the antibiotic in the medium ranges from about 10 μg / ml to about 250 μg / ml, from about 25 μg / ml to about 100 μg / ml, from about 40 μg / ml to about 65 μg / ml, Lt; / RTI >

The method may include culturing the stem cells in a medium containing serum for 10 to 350 hours before the step of differentiating into stem cell-derived skin precursor cells. The culture may be performed for 10 to 350 hours, 20 to 170 hours, or 50 to 70 hours, or 0.5 to 14 days, 1 to 7 days, or 2 to 3 days. The culture may be performed by using the isolated stem cells as P0. The number of passages of the culture is not particularly limited, and the number of passages can be appropriately selected according to the number of cells to be proliferated. The number of passages of the culture may be 1 to 20 passages, 2 to 10 passages, 3 to 7 passages, or 4 to 5 passages to obtain the required number of cumulative cells.

The method may include culturing the stem cells in a differentiation medium comprising ascorbic acid and hydrocortisone to differentiate into stem cell-derived skin precursor cells. The stem cells may be cultured in a differentiation medium containing ascorbic acid and hydrocortisone and differentiated into stem cell-derived skin precursor cells.

The term " differentiation " means that the structure or function of one another is specialized while the cells are growing by proliferation and proliferation, that is, the cells and tissues of the organism are changed in shape or function . Measuring or determining the degree of differentiation into a particular cell type may be performed by methods well known in the art. The differentiation can also be measured by measuring cell surface markers (e.g., staining cells with tissue-specific or cell-specific antibodies) using techniques such as flow cytometry or immunocytochemistry and changes in cell morphology, Changes in gene expression can be measured by examining cell morphology using an optical microscope or a confocal microscope or by using techniques well known in the art, such as polymerase chain reaction (PCR) and gene-expression profiles ≪ / RTI >

The stem cells may behave differently depending on the surrounding microenvironment such as cell division, differentiation or migration. The stem cells may be differentiated by the stimulation from the surrounding microenvironment, and the differentiated cells may be different.

The differentiation medium may be one comprising ascorbic acid and hydrocortisone. Ascorbic acid is an organic compound with antioxidant properties, which is a kind of vitamin C, is water-soluble, and has a molecular formula of C ₆ H ₈ O ₆ . The ascorbic acid may be L-ascorbic acid. The ascorbic acid may be in the form of ascorbic acid or a salt thereof. The salt of ascorbic acid may be an inorganic acid salt, an alkali metal salt or an alkaline earth metal salt, and examples thereof include inorganic acid salts such as hydrochloride, nitrate and sulfate, alkali metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt, Earth metal salt and the like, and may be L-ascorbate such as sodium L-ascorbate, magnesium L-ascorbate, potassium L-ascorbate, calcium L-ascorbate and the like. The ascorbic acid may affect proliferation and basement membrane formation of stem cell-derived skin precursor cells. The concentration of ascorbic acid in the medium may be about 0.03 μM to about 3 μM, about 0.05 to about 2 μM, about 0.1 to about 1 μM, or about 0.1 μM to about 0.5 μM.

The hydrocortisone is a kind of adrenocortical hormone and has the molecular formula C ₂₁ H ₃₀ O ₅ . The hydrocortisone may be in the form of hydrocortisone or a salt thereof. The salt of hydrocortisone may be an acid addition salt, for example, an inorganic acid salt such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, aliphatic mono- and dicarboxylate, Substituted alkanoates, hydroxyalkanoates and alkanedioates, non-toxic organic acids such as aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Hydrocortisone is a factor that stimulates the ability to differentiate into the precursor cells of the skin. It plays a role in lipid synthesis and plasma membrane formation, and can promote the formation of stratum corneum at the upper part of the epidermal layer. The concentration of hydrocortisone in the medium ranges from about 0.05 μg / ml to about 5 μg / ml, from about 0.075 μg / ml to about 3.5 μg / ml, from about 0.1 μg / ml to about 2.5 μg / About 1.25 占 퐂 / ml, about 0.3 占 퐂 / ml to about 1 占 퐂 / ml, or about 0.4 占 퐂 / ml to about 0.6 占 퐂 / ml.

For example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F- 10, F-12, DMEM / F12, a-Minimal Essential Medium, G-MEM, Iscove's Modified Dulbecco's Medium, MacCoy's 5A medium, AmnioMax complete medium, AminoMaxII complete medium , EBM-2 Basal medium, Chang's Medium, and MesenCult-XF. The differentiation medium may be one comprising serum. The serum may be fetal bovine serum (FBS), bovine calf serum (BCS), or a combination thereof. The serum may be about 1% to about 50%, about 2% to about 25%, about 5% to about 20%, or about 7.5% to about 12.5% of the total volume of the differentiation medium.

The method comprises culturing the stem cells in a differentiation medium for 120 to 600 hours, 150 to 500 hours, 180 to 450 hours, or 200 to 300 hours, or 5 to 25 days, 6 to 21 days, 7 to 18 days, 8 to 15 days, Day, or 9 to 12 days, to differentiate into stem cell-derived skin precursor cells.

The stem cell-derived skin precursor cells may have a limited ability to differentiate from the stem cells in terms of their differentiation potential and self-renewal ability. The stem cell-derived skin precursor cells are selected from the group consisting of keratin 5 (Krat5), keratin 1 (Krt1) and keratin 14 (Krt14) in comparison with stem cells before culturing in the differentiation medium Or more. That is, as the stem cells differentiate into precursor cells of the skin, expression of at least one selected from the group consisting of Krt5, Krt1, and Krt14 may be increased. The stem cell-derived skin precursor cells may be differentiated into a state in which a large amount of Krt10 and / or IVL is not reached in the late stage of differentiation depending on the degree of differentiation. The increase may be that the expression of the gene, that is, the amount of the mRNA and / or the amount of the protein is increased more than 2 times, more than 4 times, more than 8 times, more than 10 times, or more than 20 times.

The method may include culturing the differentiated stem cell-derived skin precursor cells in a medium to produce a culture of stem cell-derived skin precursor cells. The culturing can be carried out by culturing the differentiated stem cell-derived skin precursor cells in a medium without subculturing. The medium is a medium suitable for protein production, and refers to a medium capable of producing a useful protein and secreting it to the outside of the cell through interaction between cells during the culture of stem cell-derived precursor cells. The medium may be a serum-free medium. The medium may be a medium that does not contain choline chloride, phenol red (or phenol sulfopthalene), or a combination thereof. For example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10 , F-12, DMEM / F12,? -MEM (alpha-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax complete medium, EBM-2 Basal medium, Chang's Medium, and MesenCult-XF.

The method comprises culturing the differentiated stem cell-derived skin precursor cells in a medium for 10 to 350 hours, 15 to 200 hours, 20 to 170 hours, or 40 to 80 hours, or 0.5 to 15 days, 1 to 8 days, 1.5 to 4 Day, or 2 to 3 days to produce a culture of stem cell-derived skin precursor cells.

The culture of stem cell-derived skin precursor cells may be a medium, a differentiated skin precursor cell, or a mixture thereof obtained during or after culturing the differentiated stem cell-derived skin precursor cells in a medium .

The differentiated stem cell-derived skin precursor cells can be secreted out of the cell precursor cell protein through intercellular interactions while being cultured in a medium. The stem cell-derived skin precursor cells may vary in gene expression pattern by stimulation from the surrounding environment, and thus the types and amounts of the proteins secreted from stem cell-derived skin precursor cells may vary. The stimulation may include a culture medium in which the stem cell-derived skin precursor cells are cultured and / or a culture time. That is, the kind and amount of the protein secreted from the stem cell-derived skin precursor cells may vary depending on the medium to be cultured and / or the culture time, and the composition and content of proteins present in the stem cell-derived precursor cell culture fluid may vary .

The method may include recovering the stem cell-derived skin precursor cell culture from the culture of the stem cell-derived skin precursor cells. The recovery may be to obtain the culture of the stem cell-derived precursor cells or the supernatant thereof obtained during or after culturing the differentiated precursor cells in the medium. The recovery is performed by centrifuging the culture of the stem cell-derived precursor cells or the supernatant thereof obtained during or after culturing the differentiated stem cell-derived skin precursor cells in a medium, Cell-derived skin precursor cells and / or macromolecules.

The stem cell-derived skin precursor cell culture fluid, pharmaceutical composition and wound are as described above.

Another aspect provides a method of treating a wound in an individual comprising administering the pharmaceutical composition to a subject.

Administration can be by any method known in the art. Administration may be by any means, either directly or indirectly, by any means, such as by intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration, ≪ / RTI > The administration can be systemically or locally administered. The administration may be topical administration to the site where the wound is present. The administration may be by, for example, application or injection. The application refers to any method of contacting the composition to the skin of a subject in any suitable manner, thereby allowing the composition to be absorbed into the skin.

The subject may be a mammal, such as a person, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The entity may be an entity that requires wound healing. The subject may be an individual in need of promoting collagen synthesis.

The method may further comprise adding to the composition a composition comprising from about 0.01% to about 60%, from about 0.02% to about 50%, from about 0.05% to about 30%, from about 0.1% to about 20%, from about 0.2% to about 19% % To about 17%, about 0.5% to about 16%, about 0.6% to about 15%, about 0.8% to about 14%, about 1% to about 13%, about 2% to about 12% A composition comprising a stem cell derived precursor cell culture medium of about 10%, about 4% to about 7%, about 4% to about 6%, or about 5% 5 days to 150 days, 10 days to 75 days, 15 days to 50 days, 20 days to 40 days, or 25 days to 35 days, more than 1 month, more than 1 month, more than 3 months, Day, or more than once a day.

The method comprises administering a stem cell derived precursor cell culture broth or concentrate thereof to a subject in an amount of about 0.03 ml / kg to about 500 ml / kg, about 0.06 ml / kg to about 250 ml / kg, about 0.15 ml / kg to about 100 ml kg, about 0.3 ml / kg to about 50 ml / kg, about 0.6 ml / kg to about 25 ml / kg, about 1.5 ml / kg to about 10 ml / kg. < / RTI >

According to the composition for treating wounds according to one aspect, skin regeneration of an individual can be promoted. According to the method for producing a pharmaceutical composition for treating wounds according to one aspect, even when the above steps are repeatedly performed, the composition of the protein useful for the treatment of wounds containing stem cell- The composition can be obtained, and the wound healing of the individual can be promoted.

FIG. 1 shows the results of differentiation of skin derived undifferentiated keratinocytes into epidermal cells, and differentiation of placenta-derived stem cells and umbilical cord stem cells into skin precursors for 11 days in differentiation medium, Fig.
FIG. 2 is a graph showing the results of differentiation of the placenta-derived stem cells from the differentiation medium to the skin precursor cells at 3 days, 5 days, 7 days, 9 days, 11 days, 13 days, 15 days, 17 days, 19 days, and 21 days , Krt5, Krt1, IVL, Krt14, and Krt10.
FIGS. 3 and 4 show the results of differentiation of the placenta-derived stem cells from the differentiation medium to the skin precursor cells at 3 days, 5 days, 7 days, 9 days, 11 days, 13 days, 15 days, 17 days, 19 days, Day, the result of measuring the protein level of Krt14.
Fig. 5 shows the top 22 species among cytokines with high expression levels in undifferentiated stem cell culture fluid.
FIG. 6 shows the top 24 species among cytokines having a high expression level in a stem cell-derived skin precursor cell culture solution.
FIG. 7 shows the concentrations of the kidney-1 in the stem cell-derived skin precursor cell culture medium (differentiation medium), undifferentiated stem cell culture medium (undifferentiated culture medium), and normal epidermal cell culture medium (epidermal cell culture medium).
FIGS. 8 and 9 show the result of measuring the degree of wound healing after administering the composition containing the differentiated precursor cell culture fluid to a wound-induced animal model.
Fig. 10 shows the surface antigen characteristics of placenta-derived stem cells.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: stem cell origin Skin precursor cell  Production of culture solution and confirmation of wound healing effect

1. stem cell origin Skin precursor cell  Culture preparation

(1) from placenta-derived stem cells Into skin progenitor cells  differentiation

We received informed consent from normal healthy pregnant women in advance and separated umbilical cord from placenta collected during normal placental delivery. The separated placenta and umbilical cord were each washed 2 to 5 times with Dulbecco's Phosphate-Buffered Saline (DPBS) free of Ca / Mg to remove blood. The placental tissue was then cut to a size of 1 to 5 mm. The umbilical cord was removed and the umbilical cord was cut to a size of 1 to 5 mm. Then, each of the placenta and umbilical cord was adhered to a culture vessel and cultured for 10 to 15 days. After confirming that the cells spread out from the cultured tissue, 200 U / ml of collagenase I was added for 5 to 6 hours To isolate placenta-derived stem cells and umbilical cord-derived stem cells, respectively.

To determine whether placenta - derived stem cells exhibit the characteristics of mesenchymal stem cells, flow cytometry analysis was performed for surface protein analysis. Cells were washed with DPBS and incubated with DPBS containing 2% FBS to induce CD44, CD73, CD90, CD105, CD45, CD34, CD31, CD29, CD49, CD9, HLA- The reaction was carried out for 20 minutes. Subsequently, surface antigen characteristics were analyzed by flow cytometry (FACS Calibur, Becton Bickinson). FIG. 10 shows the surface antigen characteristics of placenta-derived stem cells. As shown in FIG. 15, since the expression levels of CD44, CD73, CD90, CD105, CD29, CD49, CD9 and HLA-ABC were high in placenta-derived stem cells, . ≪ / RTI >

The separated cells were inoculated into a multi-flask at a concentration of 100 to 5,000 cells / cm ² , and the cells were cultured in the presence of 25 ng / ml of fibroblast growth factor-4 (PS-CM) medium containing 1 μg / ml of heparin, 50 μg / ml of gentamycin, and 10% of fetal bovine serum (FBS) Lt; 0 > C, 5% CO ₂ for 2 days.

Thereafter, to the cultured cells were added DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) medium, 0.3 μM ascorbic acid, 0.5 μg / ml hydrocortisone and 10% The differentiation medium containing serum (Fetal Bovine Serum: FBS) was added to a multi-flask at a concentration of 5 ml / cm ² and cultured at 37 ° C under 5% CO ₂ for 11 days. The control group was cultured in a medium containing no ascorbic acid and hydrocortisone.

FIG. 1 is a diagram showing the morphology of each cell after differentiation of epidermal cells into undifferentiated epidermal cells from the skin, and differentiation of placenta-derived stem cells and umbilical cord-derived stem cells into skin precursors for 11 days in a differentiation medium. Placenta - derived stem cells and umbilical cord - derived stem cells differentiated into small, uniform - sized circular cell types similar to those of skin precursor cells in the differentiation medium. As shown in Fig. 1, placenta-derived stem cells and umbilical cord-derived stem cells show the shape of skin precursor cells after being cultured in the differentiation medium.

(2) differentiated In skin progenitor cells  Confirm gene expression

(2.1) Real-time polymerase chain reaction (real-time PCR : RT- PCR ) analysis

The expression levels of the genes Krt5, Krt1, IVL, Krt14, and Krt10 expressed in the middle and late stages were measured by RT-PCR during the process of differentiating the placenta-derived stem cells of the above (1) into the skin precursor cells.

Cells were obtained at 3 days, 5 days, 7 days, 9 days, 11 days, 13 days, 15 days, 17 days, 19 days and 21 days during the process of differentiating placental stem cells into skin precursor cells. / Chloroform was used to extract RNA from the cells. The extracted RNA was reverse transcribed to synthesize cDNA. The gene expression level of the cDNA was analyzed by RT-PCR on an Applied Biosystems 700 sequence detection system (Foster City, CA, USA). At this time, a primer set, 2 x TaqMan master mixture and 20 x premade TaqMan gene expression analysis (Applied Biosystems) kit specific for the synthesized cDNA, Krt5, Krt1, IVL, Krt14 and Krt10 described in the following Table 1 were used . The PCR conditions were as follows: 95 ° C for 10 min, 95 ° C for 15 sec, and 60 ° C for 1 min, repeated 40 times. MRNA levels of Krt5, Krt1, IVL, Krt14, and Krt10 were normalized to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels.

Gene name Forward primer Reverse primer Krt5 GTC TCG CCA GTC AAG TGT GT
(SEQ ID NO: 1) GAC ACG GAG GTG AAG CTG
(SEQ ID NO: 2) Krt1 GGG TGG TTA TGG TCC TGT CT
(SEQ ID NO: 3) GGA TCT CAG GGT CAA TCT CC
(SEQ ID NO: 4) IVL CCA GGT CCA AGA CAT TCA AC
(SEQ ID NO: 5) ACT GCG GGT GGT TAT TTA TG
(SEQ ID NO: 5) Krt14 GAG CAG CAG AAC CAG GAG T
(SEQ ID NO: 7) GAG AAC TGG GAG GAG GAG AG
(SEQ ID NO: 8) Krt10 ACT ACT CTT CCT CCC GCA GT
(SEQ ID NO: 9) TGA GCT AAA TCC TCC ACC AA
(SEQ ID NO: 10)

FIG. 2 is a graph showing the results of differentiation of umbilical cord stem cells into the skin progenitor cells at 3 days, 5 days, 7 days, 9 days, 11 days, 13 days, 15 days, 17 days, 19 days, The expression levels of Krt1, IVL, Krt14, and Krt10 genes were measured. The Y axis represents the expression level of the gene normalized with human GAPDH. As shown in FIG. 2, the mRNA level of Krt14 expressed in the mid-stage in the differentiation stage of the skin progenitor cells increased after 5 days as the differentiation progressed.

(2.2) Immunoblot : IB )

During the process of differentiating the placenta-derived stem cells of (1) into precursor cells of the skin, the protein level of Krt14, a gene expressed in the middle stage, was measured by immunoblot.

Cells were obtained at 5 days, 7 days, 9 days, 11 days, 13 days, 15 days, 17 days, 19 days, and 21 days during the process of differentiating placental stem cells into skin precursor cells. And dissolved in cell lysis buffer. The cell lysis buffer used was a RIPA buffer (Thermofisher) and a protease inhibitor cocktail (Roche, Inc., Indianapolis, IN, USA). Proteins contained in the cell lysate were quantitated and 20 μg of protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for each test group and control group. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). The PVDF membrane was reacted with the primary antibody overnight at 4 ° C. On the next day, the PVDF membrane was washed with TBST solution containing Tween, Tris, and NaCl, and reacted with HRP-conjugated secondary antibody at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit system (EMD Millipore).

FIGS. 3 and 4 show the results of differentiation of the placenta-derived stem cells from the differentiation medium to the skin precursor cells at 3 days, 5 days, 7 days, 9 days, 11 days, 13 days, 15 days, 17 days, 19 days, Day, the result of measuring the protein level of Krt14. As shown in FIGS. 3 and 4, it can be seen that the amount of Krt14 protein gradually increased from 5 days after culturing the placenta-derived stem cells in the differentiation medium. On the other hand, the placenta - derived stem cells were not expressed in the ascorbic acid and hydrocortisone - containing medium, and then Krt14 gene was not expressed or expressed to a very low extent.

(3) Preparation of stem cell-derived skin precursor cell culture

(1) of the present invention.

(1), the differentiation medium was removed from the precursor cells and the remaining serum was removed by washing with DPBS. Thereafter, DMEM / F12 medium containing no choline chloride and phenol red was added to the culture plate at a rate of 2 to 3 ml / cm ² , and cultured at 37 ° C in 5% CO ₂ Lt; / RTI > for 3 days. The supernatant was then recovered from cultures of differentiated skin precursor cells and skin precursor cells mixed with media. Then, the supernatant was filtered through a 0.22 mu m filter to obtain a stem cell-derived precursor cell culture solution. In the control group, cultured placenta-derived stem cells (hereinafter referred to as undifferentiated stem cells) and normal epidermal cells cultured in a medium containing no ascorbic acid and hydrocortisone were used, and the culture medium recovered from the medium was used.

(4) stem cell origin Skin precursor cell  Analysis of components of culture media

The components of the cell-derived proteins in the differentiated stem cell-derived skin precursor cell cultures and the undifferentiated stem cell cultures obtained in (3) were confirmed by the secretory analysis. Specifically, a total of 507 cytokines were identified.

Seqrimon analysis was performed using a RayBio (TM) C-Series Human Cytokine Array (RayBiotech, Norcross, GA) according to the protocol of a RayBio Human Cytokine Antibody Array. . The relative intensity of the obtained spot was measured by Image J and corrected by background subtraction. The results were expressed as the average value of the two readings.

(4.1) Undifferentiated stem cell culture fluid Seckley Tom  analysis

Fig. 5 shows the top 22 species among cytokines with high expression levels in undifferentiated stem cell culture fluid. Table 2 shows six cytokines that are expressed alone in undifferentiated stem cell culture fluids. The undifferentiated stem cell culture medium contains the CC chemokine receptor type 4 (CCR4), the granulocyte chemotactic protein-2 (GCP-2) / chemokine (CXC motif) ligand 6 6: CXCL6), Endocan, P-selectin, CC chemokine receptor type 5 (CCR5), Regulated on Activation (Normal T Cell Expressed and Secreted: RANTES ) And a glial cell line-derived neurotrophic factor (GDNF).

No. Cytokine Signial intensity Undifferentiated culture liquid Differentiation medium One CCR4 107,943 One 2 GCP-2 / CXCL6 58,109 One 3 Endocan 57,883 One 4 P-selectin (P-selectin) 34,935 One 5 RANTES 24,995 One 6 GDNF 21,206 One

(4.2) Differentiated stem cell origin Skin precursor cell  Culture solution Seckley Tom  analysis

FIG. 6 shows the top 24 species among cytokines having a high expression level in differentiated precursor cell culture media. Table 3 shows 24 kinds of cytokines that are expressed in large amounts in differentiated precursor cell culture media. The differentiated precancerous cell culture medium contains thrombospondin (TSP), a tissue inhibitor of metalloproteinases 1 (TIMP1), a tissue inhibitor of metalloproteinases 2 (TIMP2 ), Ectodysplasin-A2 (EDA-A2) X-linked ectodysplasin-A receptor (XEDAR), Angiopoietin-1 , Secreted protein acidic and rich in cysteine (SPARC), epithelial growth factor-like and two polystatin-like domain 1 transmembrane proteins with EGF-like and two Follistatin-like domains Insulin-like growth factor-binding protein-3 (IGFBP-3), thrombose (IGFBP-3) Thrombospondin-2, tumor necrosis factor-related activity-induced cytokines (TNF-related acti vascular-induced cytokine (TRANCE), and interleukin-15 receptor alpha (IL-15R alpha).

No. Cytokine Signal strength One Trombospondin (TSP) 5,886,805 2 IGFBP-rpl / IGFBP-7 3,970,222 3 TIMP-2 3,950,540 4 EDA-A2 3,938,694 5 XEDAR 1,572,912 6 Angiopoietin-1 1,167,638 7 SPARC 1,020,366 8 GDF-3 1,009,469 9 sFRP-4 999,123 10 GRO 963,242 11 MIP 2 865,819 12 TIMP-1 785,939 13 Potential TGF-beta bp1 554,608 14 CV-2 / cross vainless -2 475,676 15 IL-6 441,917 16 TMEFF1 / Tomorogulin-1 430,143 17 Nidogen-1 413,625 18 Smad 4 391,810 19 Activin C 228,189 20 IGFBP-3 182,444 21 Thrombospondin-2 112,674 22 TRANCE 100,065 23 Activin A 76,102 24 IL-15 R alpha 51,270

(5) stem cell origin Skin precursor cell  Analysis of protein concentration in culture medium

It was confirmed that the protein of Table 3 was secreted in the differentiated pre-progenitor cell culture fluids, and their secretion amount was quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA). The control group was prepared by adding medium to placenta-derived stem cells (hereinafter, undifferentiated stem cells) cultured in a medium containing no ascorbic acid and hydrocortisone, culturing for 2 to 4 days, culturing the recovered culture medium, and adding medium to the epidermal cells And cultured for 2 to 4 days to use the recovered culture. The ELISA kit was prepared by ELISA (ELISA) for ELISA, ILH-Nidogen1 (Raybiotec, Norcross, GA) for Nidogen-1, MBS262463 (Mybiosource) for TRANCE, ELH -IL15RA (Raybiotec, Norcross, GA) was used and performed according to the manufacturer's instructions. Absorbance was measured at 450 nm using a microplate reader Epoch (BioTek Inc.) and analyzed using Gen5 (2.00) software.

Table 4 below shows the results of measurement of the concentrations of thrombospondin, TRANCE, and IL-15R alpha in differentiated precursor cell cultures. It can be seen that the differentiated precancerous cell culture medium contains a large amount of kidogen, thrombospondin, and the like. Fig. 7 shows the concentrations of kidogen-1 in differentiated skin precursor cell cultures, undifferentiated stem cell cultures, and conventional epidermal cell cultures. As shown in Fig. 7, it can be seen that the differentiated pre-adipocyte cultures have a higher than 10-fold higher content of kidogen-1 than the undifferentiated stem cell cultures and epidermal cell cultures.

No. Cytokine Concentration (pg / ml) Signal strength One Thrombospondin 63.65 5,886,805 2 Nidogen-1 15180 413,625 3 TRANCE 29 100,065 4 IL-15R alpha 19 51,270

2. stem cell origin Skin precursor cell  Confirmation of skin regeneration and wound healing promotion effect

(1) stem cell origin in the diabetic induction model Skin precursor cell  Confirmation of wound healing promoting effect of culture solution

1 showed the wound healing efficacy in a diabetic animal model. All procedures were carried out with the approval of the School of Medicine and the Commission.

1 were centrifuged at 3220 xg and at 4 캜 until the volume was concentrated to about 10 times. At this time, an amicon (Millipore, Mass., USA) column and a centrifuge 5810R (Eppendorff, Ha mburg, Germany) swing bucket were used.

Streptozotocin (Sigma) was dissolved in 0.1 M citrate buffer (pH 4.5), and dissolved streptozotocin was added to Balb / c mice at 6 to 8 weeks of age at 55 mg / kg for 5 consecutive days I was injected to induce diabetes. Serum glucose levels were then measured in mice using an Accu-Check Advantage Glucometer (Roche, Indianapolis, IN). Diabetes mellitus was diagnosed and classified when blood plasma glucose level was over 300 mg / dl.

Mice that had been confirmed to have been induced with diabetes and control mice that did not induce diabetes were anesthetized with isoflurane and treated using standard skin biopsy punches (0.5 cm in diameter, Acuderm Inc., Fort Lauderdale, FL, USA) The entire skin layer was removed to cause skin wounds on the dorso-lateral side. The upper and lower wounds were about 2.5 cm. Immediately after the operation, each of the basic medium, the differentiated placental stem cell-derived skin precursor cell liquid concentrate, and the undifferentiated placenta stem cell culture liquid concentrate was repeatedly administered to the mice at a total of two times at 100 μl / mouse every 3 days. The epidermal growth factor (EGF) (1 μg / mouse), a major protein component of diabetic foot ulcer treatment (Daewoong Pharma, Easydew, Easyef Ointment), was mixed with a concentrate of differentiated placental stem cell- , And the mice were repeatedly administered with 100 [mu] l / mouse for 2 times at 3-day intervals.

The open wound area was measured after 0, 3, 6, 9, 12 and 15 days of treatment using known methods (Stem cells, 23: 1571-1578; 2005.). Wound images were analyzed using Image-Pro Plus (Media Cybernetics, Inc., Silver Spring, MD, USA), calculating pixel area and tracking image margins. The number of pixels was normalized to the scale of the scales on which each wound was photographed. Measurements were repeated and open wound area was expressed as a percentage of the area of the wound site at each time point divided by the area of the wound at day 0.

FIGS. 8 and 9 show the result of measuring the degree of wound healing after administering a composition containing the differentiated stem cell-derived skin precursor cell culture liquid to a wound-induced animal model. In FIGS. 8 and 9, STZ induced-DM represents a diabetic induction model, and Non-DM represents a model that did not induce diabetes. Pla-CM represents the group to which the undifferentiated placenta stem cell culture medium was administered, D-Pla-CM represents the group to which differentiated placenta stem cell-derived precursor cell culture medium was administered, D-Pla-EGF expresses the differentiated placenta stem cell (Sigma Aldrich customized product, 86310C-1000ml), and SAFC represents the group to which the base medium (Sigma Aldrich customized product, 86310C-1000ml) was administered. As shown in Figs. 8 and 9, when the composition containing the culture medium was administered to a wound-induced animal model, the wound area was significantly reduced as compared with the control group. On the ninth day after administration, the wound reduction rate of the placenta stem cell-derived skin precursor cell culture group was about 38% faster than that of the undifferentiated placenta stem cell culture group. It was confirmed that the precursor cell culture derived from differentiated stem cells can heal wound faster than the undifferentiated stem cell culture solution.

<110> CHA BIOTECH CO., LTD. <120> Composition for          epidermal stem cell conditioned medium and use thereof <130> PN119665 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Krt5 primer <400> 1 gtctcgccag tcaagtgtgt 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Krt5 primer <400> 2 gacacggagg tgaagctg 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Krt1 primer <400> 3 gggtggttat ggtcctgtct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Krt1 primer <400> 4 ggatctcagg gtcaatctcc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IVL primer <400> 5 ccaggtccaa gacattcaac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IVL primer <400> 6 actgcgggtg gttatttatg 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Krt14 primer <400> 7 gagcagcaga accaggagt 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Krt14 primer <400> 8 gagaactggg aggaggagag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Krt10 primer <400> 9 actactcttc ctcccgcagt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Krt10 primer <400> 10 tgagctaaat cctccaccaa 20

Claims (10)

A stem cell-derived skin precursor cell culture fluid. The pharmaceutical composition according to claim 1, wherein the wound is wound, abrasion, bruise, staple, rolled, or lath. The pharmaceutical composition according to claim 1, wherein the stem cell is an embryonic stem cell, an adult stem cell, or a degenerated stem cell. The pharmaceutical composition according to claim 1, wherein the stem cell is a mesenchymal stem cell isolated from umbilical cord, placenta, fat, bone marrow, umbilical cord blood or amniotic fluid. [3] The stem cell-derived skin precursor cell culture solution according to claim 1, wherein the stem cell derived precursor cell culture solution is selected from the group consisting of Thrombospondin (TSP), Tissue inhibitor of metalloproteinases 1 (TIMP1) TEDA, Tissue inhibitor of metalloproteinases 2: TIMP2, Ectodysplasin-A2: EDA-A2, X-linked ectodysplasin-A receptor: XEDAR, Angiopoietin-1, secreted protein acidic and rich in cysteine (SPARC), epithelial growth factor-like and two transmembrane proteins with EGF -like and two Follistatin-like domains 1 / Tomoregulin-1: TMEFF1 / Tomoregulin-1, Nidogen-1, Insulin- like growth factor- : IGFBP-3), Thrombospondin-2, Tumor Necrosis Factor-Related A pharmaceutical composition comprising at least one selected from the group consisting of sex derived cytokines (TNF-related activation-induced cytokine:: TRANCE), and IL-15 receptor alpha (IL-15R alpha interleukin-15 receptor alpha). [3] The stem cell-derived skin precursor cell culture solution according to claim 1, wherein the stem cell derived precursor cell culture solution is selected from the group consisting of Thrombospondin (TSP), Tissue inhibitor of metalloproteinases 1 (TIMP1) TEDA, Tissue inhibitor of metalloproteinases 2: TIMP2, Ectodysplasin-A2: EDA-A2, X-linked ectodysplasin-A receptor: XEDAR, Angiopoietin-1, secreted protein acidic and rich in cysteine (SPARC), epithelial growth factor-like and two transmembrane proteins with EGF -like and two Follistatin-like domains 1 / Tomoregulin-1: TMEFF1 / Tomoregulin-1, Nidogen-1, Insulin- like growth factor- : IGFBP-3), thrombospondin-2, tumor necrosis factor At least one concentration selected from the group consisting of TNF-related activation-induced cytokine (TRANCE), and interleukin-15 receptor alpha (IL-15R alpha) &Lt; / RTI &gt; [Claim 2] The pharmaceutical composition according to claim 1, wherein the stem cell-derived skin precursor cell culture fluid is 0.01 to 60% of the total volume of the cosmetic composition. The stem cell-derived skin precursor cell culture solution according to claim 1,
Culturing the stem cells in a differentiation medium containing ascorbic acid and hydrocortisone to differentiate into stem cell-derived skin precursor cells;
Culturing the differentiated stem cell-derived skin precursor cells in a medium to produce a culture of stem cell-derived skin precursor cells; And
A skin cell precursor cell culture solution derived from a stem cell, a concentrate thereof, or a lyophilized product obtained from the culture of a skin cell precursor cell culture derived from a stem cell from a culture of the stem cell-derived skin precursor cell; Composition. Culturing the stem cells in a differentiation medium containing ascorbic acid and hydrocortisone to differentiate into stem cell-derived skin precursor cells;
Culturing the differentiated stem cell-derived skin precursor cells in a medium to produce a culture of stem cell-derived skin precursor cells; And
And recovering the stem cell-derived precursor cell culture liquid from the culture of the stem cell-derived skin precursor cells. A method of treating a wound in an individual comprising administering to the subject a composition of any one of claims 1-6.

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